Engineering Protein Stability and Specificity Using Fluorous Amino Acids: The Importance of Packing Effects

Abstract

The incorporation of extensively fluorinated, or fluorous, analogues of hydrophobic amino acids into proteins potentially provides the opportunity to modulate the physicochemical properties of proteins in a predictable manner. On the basis of the properties of small fluorocarbon molecules, extensively fluorinated proteins should be both more thermodynamically stable and self-segregate through "fluorous" interactions between fluorinated amino acids. We have examined the effects of introducing the fluorous leucine analogue l-5,5,5,5',5',5',-hexafluoroleucine (hFLeu) at various positions within the hydrophobic core of a de novo-designed four-alpha-helix bundle protein, alpha(4). The stabilizing effect of hFLeu is strongly dependent on the positions at which it is incorporated, with per-residue DeltaDeltaG(degrees)((fold)) ranging from -0.09 to -0.8 kcal mol(-1) residue(-1). In particular, incorporating hFLeu at all the "a" positions or all the "d" positions of the hydrophobic core, thereby creating an alternating packing arrangement of leucine and hFLeu, leads to very stably folded proteins that exhibit higher per-residue DeltaDeltaG(degrees)((fold)) values than proteins that are packed entirely with hFleu. We conclude that efficient packing of the fluorous amino acid within the hydrophobic core provides a more important contribution to enhancing protein stability than do fluorocarbon-fluorocarbon interactions between fluorinated protein side chains.

Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

“…Although they each contain the same number of hFLeu residues, a 4 F 3 (6-13) and a 4 F 3 (17)(18)(19)(20)(21)(22)(23)(24) are significantly less stable than a 4 F 3 a and a 4 F 3 d. These differences may be explained by vertical packing interactions of the ''b-e'' and ''c-g'' interfaces, the importance of which has been highlighted in studies on other antiparallel coiled-coil proteins. [38][39][40] The knobs-into-holes packing of ''b-e'' and ''c-g'' interfaces for a 4 F 3 a and a 4 F 3 d contain only like residues, in which Leu residues pack exclusively into the vertical hole created by two Leu residues of the adjacent helix and hFLeu residues follow a similar packing arrangement at the opposite interface.…”

“…21 The introduction of three hFLeu residues stabilizes the folding of a 4 F 3 d by -8.6 kcal mol À1 relative to a 4 H. For the present studies we initially synthesized three new a 4 variants: a 4 F 3 (6-13), which contains hFLeu in place of Leu at positions 6, 10, and 13; a 4 F 3 (17)(18)(19)(20)(21)(22)(23)(24), which contains hFLeu in place of Leu at positions 17, 20, and 24; and a 4 tbA 6 , which contains the leucine analog b-t-butylalanine in place of Leu at all 6 ''a'' and ''d'' positions.…”

“…We were unable to obtain welldiffracting crystals of a 4 F 3 (17)(18)(19)(20)(21)(22)(23)(24), precluding this protein from structural comparison. (Table II), with all residues being well resolved except the last residue of both the A and B chains.…”

“…The denaturation curves were fitted assuming a two-state equilibrium between unfolded monomer and folded tetramer as described previously. 21,35 Crystallization Peptides were dissolved in 10 mM Tris buffer (pH 7.0) to a concentration of 6 mM as determined by absorbance at 280 nm. Crystals were grown by vapor diffusion at 20 C in a hanging drop with 2 lL peptide and 2 lL precipitant containing 100 mM CHES buffer (pH 9.0) and 48% PEG 400 for a 4 tbA 6 , 100 mM Tris buffer (pH 7.8) and 55% PEG 400 for a 4 F 3 d, and 100 mM Tris buffer (pH 8.5) and 48% PEG 600 for a 4 F 3 (6)(7)(8)(9)(10)(11)(12)(13).…”

“…[12][13][14] However, our laboratory and others have focused on introducing highly fluorinated analogs of residues such as valine, leucine, isoleucine, and phenylalanine into proteins as a means of enhancing stability. 12,[15][16][17][18][19][20][21][22][23] In most cases, these modified proteins display significantly increased stability toward unfolding by chemical denaturants and organic solvents, as well as increased resistance to proteolytic degradation. 20,[24][25][26] Currently, our understanding of how fluorination stabilizes protein folding is impeded by the lack of structures for proteins containing highly fluorinated amino acids.…”